U.S. patent number 8,597,944 [Application Number 13/172,027] was granted by the patent office on 2013-12-03 for culture systems for ex vivo development.
This patent grant is currently assigned to Advanced Cell Technology, Inc.. The grantee listed for this patent is Karen B. Chapman, Irina V. Klimanskaya, Michael D. West. Invention is credited to Karen B. Chapman, Irina V. Klimanskaya, Michael D. West.
United States Patent |
8,597,944 |
West , et al. |
December 3, 2013 |
Culture systems for ex vivo development
Abstract
The present invention provides methods for the culture of animal
pluripotent stem cells and their differentiated progeny cells,
tissues, and organs, and nonhuman animal embryos and fetuses.
Inventors: |
West; Michael D. (Mill Valley,
CA), Chapman; Karen B. (Mill Valley, CA), Klimanskaya;
Irina V. (Upton, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
West; Michael D.
Chapman; Karen B.
Klimanskaya; Irina V. |
Mill Valley
Mill Valley
Upton |
CA
CA
MA |
US
US
US |
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Assignee: |
Advanced Cell Technology, Inc.
(Worcester, MA)
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Family
ID: |
34798830 |
Appl.
No.: |
13/172,027 |
Filed: |
June 29, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110256622 A1 |
Oct 20, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11478780 |
Jun 29, 2006 |
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PCT/US2005/000103 |
Jan 3, 2005 |
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60534447 |
Jan 2, 2004 |
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60539796 |
Jan 28, 2004 |
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Current U.S.
Class: |
435/366;
435/373 |
Current CPC
Class: |
C12N
15/873 (20130101); C12N 5/0606 (20130101); C12N
2502/04 (20130101) |
Current International
Class: |
C12N
5/071 (20100101); C12N 5/00 (20060101) |
Field of
Search: |
;435/366,373 |
References Cited
[Referenced By]
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|
Primary Examiner: Ton; Thaian N
Attorney, Agent or Firm: Mittler; E. Stewart
Parent Case Text
This application is a continuation of U.S. application Ser. No.
11/478,780, filed on Jun. 29, 2006, which is a continuation of PCT
Application Ser. No. PCT/US2005/00103, filed on Jan. 3, 2005, which
claims priority to U.S. Provisional Application Ser. No.
60/534,447, filed Jan. 2, 2004 and U.S. Provisional Application
Ser. No. 60/539,796, filed Jan. 28, 2004, each of which is
incorporated herein by reference.
Claims
We claim:
1. A cell co-culture comprising human pluripotent stem cells and
chicken embryonic fibroblasts and the digest of chicken embryo
heads.
2. The co-culture of claim 1, wherein the human pluripotent stem
cells are human embryonic stem cells.
3. The co-culture of claim 1, wherein the chicken embryonic
fibroblasts are obtained from a 7-8 day old embryo.
Description
FIELD OF THE INVENTION
This invention generally relates to cells, tissue, and organ
culture technology. More particularly, the invention relates to
methods for culturing and differentiating animal pluripotent stem
cells and non-human mammalian embryos and fetuses.
BACKGROUND OF THE INVENTION
Advances in nuclear transfer and embryonic stem cell technology
have facilitated the cloning of non-human animals for diverse
applications including agriculture, xenotransplantation, disease
models, recombinant protein production, and novel means of
manufacturing human cells for use in medical therapies, diagnosis,
and discovery research. Each of these practical applications would
benefit from new technologies to improve efficiencies in the
production of animals, tissues, and cells. In the case of animal
cloning, the high cost of recipient females to gestate the cloned
fetuses often makes the commercialization of cloned animals
impractical. In the case of the therapeutic uses of pluripotent
stem cells, many pluripotent cells such as human embryonic stem
(hES) cells, are problematic to culture using traditional cell
culture technology. The cells are dependent on a close association
with similar undifferentiated cells and often require being
cultured in juxtaposition with embryonic fibroblast feeder cells in
order to maintain them in the undifferentiated state.
In addition, while some cells such as hES cells have a demonstrated
potential to differentiate into any and all of the cell types in
the human body including complex tissues, and while genes expressed
uniquely in many differentiated cell types are known allowing
genetic selection and purification of populations of any cell type
of interest, nevertheless, there is need for new technologies to
influence the differentiation of pluripotent stem cells such as hES
cells, new means of allowing the cells to differentiate in a three
dimensional tissue culture environment, and novel means of
purifying the target cells of interest, and techniques such as
these that can be performed in SPF conditions to minimize the risk
of pathogen transmission into humans.
In the field of the cultivation of human cells for human cell
therapy, regulatory agencies require production methods wherein the
cells are grown in defined conditions with stringent control over
contact of the cells (or anything that may come in contact with the
cells) with uncharacterized materials that are a potential source
of pathogens. In the case of human embryonic stem (hES) cells, it
is desirable to identify a means of cultivating the cells in
pathogen-free conditions, differentiating downstream progeny of the
cells, scale up the number of the cells for batch production,
cryopreservation, and genetic modification.
The original culture of hES cells as reported by Thomson et al
(Science. 1998 Nov. 6; 282(5391):1145-7) was accomplished by
culturing the inner cell mass of human blastocysts in co-culture
with feeder layer of embryonic murine fibroblasts under culture
conditions well known in the art of tissue culture to generate ES
cell lines. The murine fibroblasts provide largely uncharacterized
factors that promote the growth of ES cells while maintaining them
in an undifferentiated state. However, the embryonic murine
fibroblasts are also a potential source of pathogens including
uncharacterized retroviruses. Therefore, novel means of isolating,
culturing, and differentiating hES cells and other cells are of
great practical value. While avian CEFs have been shown to support
the growth of murine ES cells (Yang & Petitte, 1994), and the
use of avian cytokines has been described in non-human mammalian
embryonic stem cell culture, (Poultry Science 73: 965-974), there
has been no description of the possibility that avian CEFs could be
useful in providing SPF support for the growth of other mammalian
ES cells such as hES cells.
In addition, because of the innate capacity of hES cells to
organize into complex three dimensional tissues including
organogenesis, and because the growth of tissues in culture systems
beyond the size of approximately 0.5 mm in thickness is impractical
without a means of supplying vascular support, there is a need for
developing conditions that allow for the growth of solid tissues
and conditions that provide suitable vascular support for such
growing tissue with a dimensions of greater than 0.5 mm while
maintaining the cells in a specific pathogen-free environment.
The avian egg is a relatively well-characterized structure that has
evolved as a means of providing physiological support to a
developing vertebrate embryo, including nutritional support, waste
disposal, and gas exchange. The ovum of avian species such as the
domestic chicken (Gallus domesticus) is that part of the egg
commonly called the "yolk" (FIG. 1). The bulk of the ovum is a
colloidal suspension of nutrients while a small volume of cytoplasm
is concentrated in a region approximately 3 mm in diameter called
the blastodisc on the animal pole. Following fertilization, the
ovum traverses the oviduct acquiring albuminous material (egg
white) and finally the shell membrane and the calcified egg
shell.
In the case of an egg that has become fertilized by sperm
subsequent to ovulation and prior to encapsulation into the shell,
the blastodisc will undergo repeated rounds of karyokinesis and
cytokinesis until at about the time the egg is laid, a collection
of cells called the blastoderm has formed that is roughly
equivalent to the stage of mammalian embryos at the blastocyst
stage. Therefore, cultured avian blastodermal cells are
occasionally referred to as avian embryonic stem cells (aES cells)
and those from species of domestic chicken are referred to as
chicken embryonic stem (cES) cells (U.S. Pat. No. 5,340,740).
Following the formation of primitive germ layers of the avian
embryo proper, extraembryonic membranes begin to form that will
function to support the developing embryo. As shown in FIG. 2,
these include the splanchnopleure that will form the yolk sac, the
somatopleure, that will form the amnion and the chorion, and the
allantoic membrane, that will eventually fuse with the chorion to
form the chorioallantoic membrane. These membranes become
vascularized and provide the developing embryo with nutrients from
the yolk sac and gas exchange across the egg shell.
In contrast to avian species, mammalian development is viviparous
and often occurs in the context of the uterus, where embryonic
membranes form analogous to that in the avian egg, but the
extraction of nutrition from the maternal circulation can occur
either through either the chorion, the allantoic membrane, or the
yolk sac membrane depending on the mammalian species. Generally
speaking, in most mammals, the yolk sac provides little if any
nutritional support. The avian egg provides an unusually promising
environment for the cultivation of human cells. As described
herein, novel means of culturing and maintaining hES cells, hED
cells, and cells differentiated from such cells are described
utilizing telolecithal or eutelolecithal eggs or cells derived from
embryonated telolecithal or eutelolecithal eggs. In addition, it is
possible to utilize telolecithal or eutelolecithal eggs to support
the in ova development on non-human mammalian embryos and fetuses
and to reconstitute embryonic stem cells and embryo-derived cells
from chromatin from mammalian species.
SUMMARY OF THE INVENTION
The present invention provides methods for the culture of animal
pluripotent stem cells and their differentiated progeny cells,
tissues, and organs, and nonhuman animal embryos and fetuses.
More specifically, this invention provides a novel method of
culturing embryos, fetuses, cells, tissues, and organs in ovo in
telolecithal or eutelolecithal eggs and for the culture of hES
cells, hED cells, and cells differentiated from such cells in
co-culture with cells derived from embryonated telolecithal or
eutelolecithal eggs for numerous commercial applications that
improves yield, efficiency, cost, and risk in each of the above
categories.
In one aspect of the invention, the method comprises: the
utilization of an unfertilized telolecithal or eutelolecithal egg
of the avian or egg-laying mammal species as a culture system for
the growth and differentiation of mammalian stem cells.
In another aspect of the invention, stem cells are implanted within
the vitelline membranes of the telolecithal or eutelolecithal
oocyte and subsequently incubated to allow the differentiation of
mammalian extraembryonic membranes whereby a mammalian yolk sac
splanchnospleuric membrane surrounds the avian yolk.
In still another aspect of the invention, mammalian embryonic cells
can be injected in ovo in juxtaposition to the vitelline membrane
and incubated over time to allow the formation of a plurality of
mammalian extraembryonic membranes in the avian egg, including the
formation of mammalian splanchopleure, somatopleure, chorionic
membrane (CAM), allantoic membrane, amniotic membrane, or yolk sac
membranes. The generation of such extraembryonic membranes has
great utility in supporting the differentiation of hES or hED cells
for purposes of research or manufacture, or, in the case of
non-human mammalian species, in supporting advanced development of
embryos and fetuses for research or production of agricultural
animals.
In yet another aspect of the invention, mammalian embryonic cells
(such as hES cells or hED cells, or cells differentiated from such
cells) can be injected in ova in juxtaposition to the vitelline
membrane of an embryonated avian egg to produce differentiated
cells vascularized by the vitelline vascular plexus.
In another aspect of the invention, mammalian pluripotent stem
cells (such as hES cells or hED cells, or cells differentiated from
such cells) are injected in juxtaposition with the CAM, or in a
region of the egg in which the CAM will eventually invade. The
vasculature of the CAM then supplies vascularization to the growing
and differentiating mass of cells.
In still yet another aspect of the invention, mammalian pluripotent
stem cells are injected in the amniotic cavity, albumin, air space,
allantoic cavity, extraembryonic coelom, or the yolk sac of the egg
and allowed to differentiate.
In another aspect of the invention, inducers such as factors
including hormones, growth factors, extracellular matrix
components, or inducer cells are introduced into the avian egg with
the stem cells of the above-mentioned protocols in order to
influence the course of differentiation of the injected mammalian
pluripotent stem cells.
In a particular aspect of this invention, the inducer cells of the
pervious embodiment include avian SPF cells from diverse
differentiated cell lineages including somatic cells obtained from
the differentiation of chicken embryonic stem (cES) cells.
In another aspect of the invention, whole and intact nonhuman
embryos and fetuses can be cultured in the avian egg with or
without a shell or shell membrane (in ovo) through the injection of
nonhuman embryos or embryo-derived cells into the egg in
juxtaposition to the vitelline membrane. Whole and intact human
embryos could also be developed in ovo using the described
invention, however, it is the belief of the inventors that the use
of the technology for this purpose is not ethical and claims for
such uses are not sought in the present invention.
In still another aspect of the present invention, intact non-human
mammalian embryos and fetuses can be grown in ovo and used to
induce the differentiation of injected mammalian pluripotent stem
cells including hES and hEDC cells by injecting such hES, hED, or
cells differentiated from such cells into chosen sites of the
differentiating non-human animal embryo or fetus to induce the
differentiation of such injected cells.
In another particular aspect of the invention, nonhuman mammalian
embryos and fetuses can be cultivated in ovo by means of the
transfer of chromatin into the blastodisc of an unfertilized avian
egg, where the avian oocyte is activated and induced to undergo
rounds of karyokinesis and cytokinesis and subsequent development.
Human chromatin can also be introduced into the blastodisc of the
avian egg for the purpose of reconstituting intact embryonic cells
from reprogrammed chromatin, but the development of intact human
embryos post gastrulation and fetuses by this means is considered
unethical and claims relating to human post-gastrulation embryos or
fetuses cultured in ovo are not sought in this application.
In another aspect of the invention, embryonic cells from SPF
species including SPF embryonic chicken cells are used as feeder
cells for the in vitro cultivation of mammalian ES cells including
hES and hEDC cells in vitro or in ova.
In another aspect of the invention, somatic cells from SPF species
including SPF embryonic chicken cells are used as cells to induce
the differentiation of hES or hED cells or cells differentiated
from such cells. The SPF inducer cells may be viable or mitotically
inactivated by radiation or chemical treatment, and may be
co-cultured with the human stem cells in a variety of culture
conditions including in vitro and in ovo co-culture.
Other features and advantages of the invention will be apparent
from the following description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing illustrating the transfer of stem cells in
juxtaposition to the vitelline membrane of the unfertilized or
early embryonated avian egg. In the example shown, the cells are
injected with an inducer to influence the course of differentiation
of the stem cells.
FIG. 2 is a drawing of an artificial culture vessel for maintaining
mammalian pluripotent stem cells and derivative cells in the
presence of components of a telolecithal or eutelolecithal egg.
FIG. 3 is a drawing of the various anatomical structures of the
fertilized chicken egg, showing the location of the chorioallantoic
membrane (CAM) and the placement of mammalian pluripotent stem
cells and inducer in juxtaposition to the CAM membrane.
FIG. 4 is a drawing showing the result of placement of mammalian ES
cells or embryo-derived cells within the vitelline membrane of an
embryonated egg such that the growing teratoma is vascularized by
the chick's vitelline vascular plexus.
FIG. 5 shows an hematoxylin-and-eosin stained tissue section from a
human teratoma formed by the placement of human ES cells within the
vitelline membrane of an embryonated egg.
FIG. 6 shows the use of SPF chick embryo fibroblasts to stably
maintain hES cell lines in an undifferentiated state. Morphology
(A-C) and markers (D-I) of undifferentiated hES cells grown on CEF:
A--colonies of hES cell line H9 on CEF. B, C--colonies of the hES
cell line H1 grown on CEF (B) vs. on MEF (C); D-I, hES cell line H7
cultured on CEF (4 passages): D, Oct-4; E, SSEA-3; F, SSEA-4; G,
alkaline phosphatase; H, TRA-1-60; I, TRA-1-81. Original
magnification: A, .times.38; B-I, .times.200
DETAILED DESCRIPTION OF THE INVENTION
TABLE-US-00001 Table of Abbreviations [Ca.sup.+2]i Intracellular
calcium concentration CAM Chorioallantoic membrane CEF Chick Embryo
Fibroblast cES Cell Chicken embryonic stem cell ES Cell Embryonic
stem cells derived from a morula or blastocyst- staged mammalian
embryo produced by the fusion of a sperm and egg cell, nuclear
transfer, parthenogenesis, or the reprogramming of chromatin and
subsequent incorporation of the reprogrammed chromatin into a
plasma membrane to produce a cell. hEDC Human Embryo-Derived Cells
hES Cell Human embryonic stem cells ICM Inner Cell Mass of the
blastocyst embryo. ICSI Intracytoplasmic sperm injection MII
Metaphase II NT Nuclear Transfer SPF Specific Pathogen-Free
The present invention provides methods for the culture of mammalian
stem cells, differentiated progeny cells, tissues and organs, and
non-human mammalian embryos and fetuses in a telolecithal or
eutelolecithal egg such as that of avian or egg laying mammalian
species (in ovo). The term "in ovo" refers to residence within a
shelled telolecithal or eutelolecithal egg, or in the presence of
the components of such an egg or eggs cultured in a container other
than an egg shell, such container being composed of polymers,
glass, or metal. The telolecithal or eutelolecithal eggs useful in
the present invention may be from the common domestic chicken
(Gallus gallus domesticus) or from any other avian species
including but not limited to the turkey (Meleagris), quail
(Coturnix), and duck (Anas) or an egg-laying mammals such as those
of the Order Monotremata. The avian eggs useful in this invention
for the production of therapeutic products include specific
pathogen-free (SPF) eggs. The term "specific pathogen-free" refers
to eggs that have been obtained from animals reared in conditions
to insure that the animals and their eggs are free of known
pathogens including avian pathogenic viruses.
The term "suicide gene" refers to genes that may be introduced into
the mammalian stem cells or into the avian inducer cells or into
the avian system providing vascular support, such that upon
stimulation, the cells that carry the suicide gene can be induced
to die. Such suicide genes are well known in the art and include
the use of herpes simplex virus thymidine kinase that in the
presence of gancyclovir can cause the death of the cell carrying
the gene.
The term "mitotically inactivated" refers to cells that have been
rendered incapable of subsequent cell division by the exposure of
such cells to agents that damage the DNA of such cells such that
the cells undergo DNA damage checkpoint arrest or apoptosis. Such
mitotic inactivation can be achieved by techniques well known in
the art such as the use of exogenous radiation, or chemical agents
including mitomycin C.
The term "teratoma" refers to a benign mass of cells
differentiating from pluripotent stem cells that organize into
complex tissues in three dimensions, though lacking the normal and
intact form of an animal and incapable of independent life. By way
of example, teratomas have been reported to occur following the
injection of hES cells into the skeletal muscle or peritoneum of
immunocompromised mice where such teratomas contain intestine,
skin, teeth, renal tissue, neuronal tissue, bone, cartilage, and so
on.
The term "chorioallantoic membrane" or "CAM" refers to the
outermost extraembryonic membrane that eventually lines the
noncellular eggshell membrane. The CAM is formed by the fusion of
the splanchnic mesoderm of the allantois and the somatic mesoderm
of the chorion. The fused doublet of allanois and chorion will
cover the entire inner surface of the egg shell by day 12.
The term "pluripotent stem cells" refers to animal cells capable of
differentiating into more than one differentiated cell type. Such
cells include hES cells, hEDCs, and adult-derived cells including
mesenchymal stem cells, neuronal stem cells, and bone
marrow-derived stem cells. Pluripotent stem cells may be
genetically modified or not genetically modified. Genetically
modified cells may include markers such as fluorescent proteins to
facilitate their identification within the egg.
The term "embryonic stem cells" (ES cells) refers to cells derived
from the inner cell mass of blastocysts or morulae that have been
serially passaged as cell lines. The ES cells may be derived from
fertilization of an egg cell with sperm or DNA, nuclear transfer,
parthenogenesis, or by means to generate hES cells with
homozygosity in the MHC region.
The term "human embryonic stem cells" (hES cells) refers to cells
derived from the inner cell mass of human blastocysts or morulae
that have been serially passaged as cell lines. The hES cells may
be derived from fertilization of an egg cell with sperm or DNA,
nuclear transfer, parthenogenesis, or by means to generate hES
cells with homozygosity in the HLA region.
The term "human embryo-derived cells" (hEDC) refer to
morula-derived cells, blastocyst-derived cells including those of
the inner cell mass, embryonic shield, or epiblast, or other
totipotent or pluripotent stem cells of the early embryo, including
primitive endoderm, ectoderm, and mesoderm and their derivatives,
but excluding hES cells that have been passaged as cell lines. The
hEDC cells may be derived from fertilization of an egg cell with
sperm or DNA, nuclear transfer, parthenogenesis, or by means to
generate hES cells with homozygosity in the HLA region.
In one embodiment of the invention, mammalian pluripotent stem
cells with or without inducer molecules or cells are injected
within and in juxtaposition to the vitelline membranes of the
unembryonated egg (FIG. 1). One mammalian pluripotent cell, or a
plurality of cells, for example, a colony of cultured mammalian
pluripotent stem cells such as ES cells, in particular hES or hEDC
cells, can be injected by techniques well known in the art, such as
incubating an egg at 37-39.degree. C. in 60% humidity, the shell
cleaned with 70% ethanol, and using a sterile syringe,
approximately 2.5 mL of albumin will be removed. This allows a
small, typically 1.5 cm.sup.2 window in the shell to be made and
cells to be injected with a glass pipette, and subsequent covering
the windowed portion of the shell with a sealant such as common
kitchen wrap and subsequent culture at 37.degree. C. with or
without supplemental calcium and ascorbate to approximate the
physiological levels of the corresponding mammalian species in a
standard tissue culture incubator. The egg may be injected at one
site, or multiple sites, including at or near the blastodisc,
depending on the nature of the cells and the type of product
desired.
In addition, the cells with or without inducer may be injected
within the vitelline membrane but external to the developing embryo
of an embryonated egg such that the differentiated cells are
vascularized by the vitelline vascular plexus. The differentiated
cells can then be removed from the egg and purified from the yolk
sac prior to hatching. Alternatively, the chicken can be allowed to
develop to hatching, in which case the yolk sac membrane is
absorbed within the body cavity of the chick and the mammalian
teratoma continues to develop within the body of the hatched chick
and the differentiated mammalian cells can be removed post hatch.
Some of the advantages of obtaining the cells post hatch are that
it allows more time for greater growth and development of the
teratoma and it provides early exposure of the chick to the
mammalian pluripotent stem cells which tolerizes the immune system
and lessens chances of rejection. As in the case of injection of
cells into unembryonated eggs, the injection of the cells into
embryonated eggs is by techniques well known in the art for the
injection of cells, such as the injection of avian blastodermal
cells into the blastoderm of a fertilized egg to generate chimeras.
The egg is cultured at 37.degree. C. or in the proximity to the
normal temperature for human cells (i.e. 35-39.degree. C.) at about
60% humidity, the shell cleaned with 70% ethanol, and using a
sterile syringe, approximately 2.5 mL of albumin will be removed.
This allows a small, typically 1.5 cm.sup.2 window to be made in
the shell for the introduction of cells with or without
supplemental calcium and ascorbate to approximate the physiological
levels of the corresponding mammalian species.
In another embodiment of the invention, the components of the egg
will be transferred to a container such as that shown in FIG. 2 to
replace the function of the egg shell and to facilitate the
manipulation of the culture system. Such container may contain a
transparent component to allow the viewing of the developing
tissue, ports for the removal, replacement, or addition of egg
components such as egg albumin or a culture medium or matrix
substrate substituting for albumin, egg yolk, mammalian pluripotent
stem cells including hES or hEDC, or inducer molecules or cells,
the cannulation of blood vessels within the differentiating tissue
for external circulatory or respiratory support, or a system such
as a semipermeable membrane to facilitate the diffusion of gases
and small molecules into and out of the culture system. The use of
an artificial container also allows for the introduction of egg
components from multiple eggs for culture of cells of animals of
long gestational age and where larger tissues or larger
extraembryonic membranes are desired, with or without supplemental
calcium and ascorbate to approximate the physiological levels of
the corresponding mammalian species.
In another embodiment of the invention, mammalian pluripotent stein
cells including hES and hEDC cells are injected in the proximity of
the shell membrane to form a teratoma that will subsequently become
vascularized by the growing CAM membrane (FIG. 3). Typically, in
the case of the chicken egg, the egg will be incubated at
approximately 37.degree. C. and 60% humidity, the shell cleaned
with 70% ethanol, and using a sterile syringe, approximately 2.5 mL
of albumin will be removed. This allows a small, typically 1.5
cm.sup.2 window to be cut in the shell and the shell membrane
allowing the mammalian pluripotent cells to be injected within the
albumin and in juxtaposition to the shell membrane. The mammalian
pluripotent cells may be injected between day 1 and day 17. The
teratoma may subsequently be removed and cultured in organ culture
with the attached vasculature used to perfuse the growing tissue
with blood or tissue culture media. Any residual avian cells may be
removed by activation of the avian suicide genes.
In another embodiment of the invention, mammalian pluripotent stem
cells including hES and hEDC are injected by the above techniques
in the amniotic cavity, albumin, air space, allantoic cavity,
extraembryonic coelom, or the yolk sac of the egg and allowed to
differentiate over time in the incubated egg.
In another embodiment of the invention, the inducer includes cells
that are derived from cells of a heterologous species, such as
chicken somatic cells inducing the differentiation of hES cells.
Such cells can be cells that normally occur in juxtaposition to the
cell of interest and include stromal cells and endothelial cells
from the organ or parenchyma of interest. The somatic inducer cells
can be obtained from a variety genotypes including SPF eggs to
reduce the risk of pathogen transmission. Such eggs are
commercially available (Charles River Laboratories) and are free of
such pathogens as Avian Adenovirouses I-III, Avian
Encephalomyelitis, Avian Influenza (Type A), Avian Nephritis Virus,
Avian Paramyxovirus Type 2, Avian Reovirus, Avian Rhinotracheitis
Virus, Avian Rotavirus, Avian Tuberculosis, Chicken Anemia Virus,
Endogenous GS Antigen, Fowl Pox, Hemophilus paragallinarum,
Infectious Bronchitis (Ark, Conn, JMK, and Mass), Infectious Bursal
Disease, Infectious Laryngotracheitis, Lymphoid Leukosis A,B,
Lymphoid Leukosis Viruses, Marek's Disease (Serotypes 1, 2, 3),
Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle Disease,
Reticuloendotheliosis Virus, Salmonella pullorum-gallinarum, and
other Salmonella species.
In another embodiment of the invention, the inducer cells are
derived from ES cells of a heterologous species. By way of
non-limiting example, the inducer cells may be cES cells
differentiated into somatic cells that function in inducing the
specific differentiation of hES cells. The cES cells can be
obtained from a variety genotypes including SPF eggs to reduce the
risk of pathogen transmission. In addition, since the cES cells can
be cultured indefinitely in an undifferentiated state, they can be
genetically modified using techniques well known in the art for
improved performance as inducer cells. Such genetic modifications
include the introduction of suicide genes that allow the
destruction of the inducer cells prior to use, modified to express
cell surface antigens that facilitate the removal of the inducer
cells by affinity methods well known in the art, or the inducer ES
cells may be modified by gene trap vectors in order to obtain ES
cell clones that express markers such as fluorescent proteins that
facilitate the purification and identification of particular
differentiated cell types as inducer cell lines.
In another embodiment of the invention, the inducer is one of a
number of extracellular signaling molecules including growth
factors, cytokines, extracellular matrix components, nucleic acids
encoding the foregoing, steroids, and morphogens or neutralizing
antibodies to such factors. Such inducers include but are not
limited to: cytokines such as interleukin-alpha A, interferon-alpha
A/D, interferon-beta, interferon-gamma, interferon-gamma-inducible
protein-10, interleukin-1-17, keratinocyte growth factor, leptin,
leukemia inhibitory factor, macrophage colony-stimulating factor,
and macrophage inflammatory protein-1 alpha, 1-beta, 2, 3 alpha, 3
beta, and monocyte chemotactic protein 1-3.
Differentiation agents according to the invention also include
growth factors such as 6kine, activin A, amphiregulin, angiogenin,
.beta.-endothelial cell growth factor, .beta.-cellulin,
brain-derived neurotrophic factor, C10, cardiotrophin-1, ciliary
neurotrophic factor, cytokine-induced neutrophil chemoattractant-1,
eotaxin, epidermal growth factor, epithelial neutrophil activating
peptide-78, erythropoietin, estrogen receptor-alpha, estrogen
receptor-beta, fibroblast growth factor (acidic and basic),
heparin, FLT-3/FLK-2 ligand, glial cell line-derived neurotrophic
factor, Gly-His-Lys, granulocyte colony stimulating factor,
granulocytomacrophage colony stimulating factor, GRO-.alpha./MGSA,
GRO-.beta., GRO-gamma, HCC-1, heparin-binding epidermal growth
factor, hepatocyte growth factor, heregulin-alpha, insulin, insulin
growth factor binding protein-1, insulin-like growth factor binding
protein-1, insulin-like growth factor, insulin-like growth factor
II, nerve growth factor, neurotophin-3,4, oncostatin M, placenta
growth factor, pleiotrophin, rantes, stem cell factor, stromal
cell-derived factor 1B, thrombopoietin, transforming growth
factor-(alpha, beta1,2,3,4,5), tumor necrosis factor (alpha and
beta), vascular endothelial growth factors, and bone morphogenic
proteins.
Differentiation agents according to the invention also include
hormones and hormone antagonists such as 17B-estradiol,
adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte
stimulating hormone, chorionic gonadotropin, corticosteroid-binding
globulin, corticosterone, dexamethasone, estriol, follicle
stimulating hormone, gastrin 1, glucagons, gonadotropin,
L-3,3',5'-triiodothyronine, leutinizing hormone, L-thyroxine,
melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary
growth hormone, progesterone, prolactin, secretin, sex hormone
binding globulin, thyroid stimulating hormone, thyrotropin
releasing factor, thyroxin-binding globulin, and vasopressin.
In addition, differentiation agents according to the invention
include extracellular matrix components such as fibronectin,
proteolytic fragments of fibronectin, laminin, tenascin,
thrombospondin, and proteoglycans such as aggrecan, heparan
sulphate proteoglycan, chontroitin sulphate proteoglycan, and
syndecan. Such extracellular matrix components may be injected at
or near the site of the injected pluripotent stem cells in a
soluble form or attached to an immobilized matrix such as a tissue
membrane or a membrane made of a synthetic polymer.
Differentiation agents according to the invention also include
antibodies to the previously-mentioned cytokines, growth factors,
hormones, and extracellular matrix components, and their
receptors.
The present invention also provides for a means of developing human
extra-embryonic membranes that can function to support the near
normal differentiation of cells from hES cells or hED cells in ova.
The injection of such human pluripotent stem cells such as hES
cells at or near the vitelline membrane of either an embryonated or
unembryonated egg by injection and subsequent incubation techniques
well known in the art and described in application above, results
in the differentiation of some of the injected cells into
extra-embryonic membranes such as human amnion, chorion, and yolk
sac, that in turn provide laboratory models of cell
differentiation, and the derivation of yolk sac hematopoietic
precursor cells, and extra-embryonic membranes useful in supporting
the growth and differentiation of such stem cells.
The present invention also provides for a means of developing
mammalian extra-embryonic membranes that can function to support
the near normal development of non-human fetuses. In particular,
such species as the domestic pig display extra-embryonic membrane
formation closely resembling that of avian species and such animals
can be gestated within the avian egg or within an artificial device
such as that shown in FIG. 2. The inner cell mass or embryonic disc
or embryonic stem cells of such non-human mammalian preimplantation
embryo or peri-implantation embryo can be grafted into or near the
blastodisc of an unfertilized avian egg, or the blastoderm of a
fertilized avian egg can be removed or inactivated and replaced by
the intact ICM or embryonic disc of a non-human mammalian embryo
with or without supplemental calcium and ascorbate to approximate
the physiological levels of the corresponding mammalian species
The present invention also provides a means of influencing the
differentiated state of cultured hES cells, hED cells, and cells
differentiated from such cells by co-culturing such cells with SPF
avian differentiated cells. SPF avian chick embryo fibroblasts,
including but not limited to chick embryo fibroblasts from SPF
embryonated eggs at nine days of culture may be isolated by
techniques well known in the art such as by removing such nine
day-old chick embryos, disaggregating the tissues, and plating the
cells in standard fibroblast growth conditions such as MEM medium
supplemented with 10% FBS or defined pathogen-free medium. hES
cells may then be serially passaged on mitotically-inactivated SPF
chick embryo fibroblasts instead of using feeder cells such as
murine embryo fibroblasts with an uncharacterized pathogen status.
The co-culture of hES cells with SPF chick embryo fibroblasts has a
clear utility in facilitating the scale up of hES cells in
pathogen-free culture conditions. The use of other specific SPF
chick cells may similarly be used where such cells are known to
cause the induction of differentiation in order to influence the
differentiation of hES, hED cells, or other downstream pluripotent
human cells. Examples of cell types that function as inducers of
differentiation are well known in the art and include mesodermal
cells such as the stromal cells from the aorta-gonadal-mesonephros
region which induce definitive hematopoiesis in pluripotent stem
cells, ectodermal cells such as the optic vesicle cells, or
mesenchymal cells from the optic vesicle that induce the
differentiation of ectodermal cells into lens cells, and endodermal
cells such as the induction of pancreatic islet cells, including
pancreatic beta cells from primitive endodermal epithelium by
pancreatic mesenchymal cells. Induction can also occur by
epithelio-stromal interactions and by the use of one germ-layer to
induce cells of another germ-layer, such as the use of dermal
mesoderm cells to induce epidermal differentiation such as hair
differentiation, mesodermally-derived cells that induce gut and
ultimately pancreatic islet cell differentiation, the mesodermal
cells of the ureteric bud that induce kidney differentiation, the
mesodermal induction of epithelium to produce pharyngeal thymus and
thyroid differentiation, liver mesenchymal cells that induce
primitive epithelium to differentiate into hepatic cords and liver
parenchyma, gut mesenchymal cells that induce primitive epithelial
cells to differentiate into gut, tracheal mesenchymal cells that
induce respiratory differentiation such as respiratory epithelium,
Such inducer cells can be removed from the corresponding region of
an SPF chick embryo by standard dissection, or isolated from SPF
chick ES cell lines utilizing genetic markers for that lineage of
cells, such as exogenous markers with exogenous promoters or using
the endogenous promoter and gene trap technology.
The present invention also provides a means of reconstituting
mammalian cells from chromatin, by removing or inactivating the
avian DNA from the blastodisc of an avian embryo using techniques
well known in the art, and replacing said genome with the haploid
or preferably the diploid genome of a mammalian cell. The mammalian
cell genome may be by way of example, human somatic cell-derived
chromatin that has been reprogrammed and condensed by exposure in
vitro to extracts or purified components from metaphase II oocytes
as is known in the art. Subsequent or at about the time of the
transfer of chromatin, the oocyte is activated such that there is
an elevation of intracellular calcium. Current strategies for the
activation of the oocyte in the absence of sperm, commonly known as
parthenogenetic activation are well known in the art, and include
chemical activation to elevate intracellular calcium concentration
followed by the down-regulation of maturation-promoting factor
(MPF), the injection of sperm extracts or purified sperm factor, or
incubation in strontium chloride. In addition, this invention
provides a novel method of activating the oocyte of a telolecithal
or eutelolecithal egg in conjunction with the transfer of chromatin
from a mammalian species, said method being the injection and
subsequent removal of a sperm, multiple sperm, or sperm heads, and
their subsequent removal. As a result of chromatin transfer and
activation, rounds of karyokinesis and cytokinesis that follow
result in cells similar in nature to hES or hEDC cells on the in
juxtaposition to the vitelline membrane as previously
described.
Applications
It is envisioned that the disclosed methods for the culture of
animal tissues are generally useful in mammalian subjects,
including human and non-human subjects, and particularly in the
culture of non-human embryos and fetuses and for the culture and
differentiation of mammalian pluripotent stem cells, in particular,
hES cells and hEDC.
Following a review of the present disclosure, one skilled in the
art of stem cell culture and the manipulation of telolecithal eggs
such as avian eggs, can readily implement the invention in the
culture of non-human mammalian embryos and fetuses, and in the
culture of mammalian stem cells including human stem cells. As
described further herein below, the methods of the present
invention can be used for culturing non-human embryos such as pigs
to advanced stages of development, and for the manufacture of
animal cells, such as human cells useful in drug discovery, basic
research, and in cell therapy.
A. Development of Mammalian Cells and Tissues in the Avian Egg
In one embodiment of the invention, hES cells and other mammalian
ES cells are cultured and differentiated within the avian egg. The
term "avian egg" refers to the fertilized or unfertilized egg of an
avian species including but not limited to eggs of the domestic
chicken (Gallus domesticus), the turkey, duck, ostrich, and quail.
However, complex tissues can be produced using this invention,
similar to the production of teratomas which are disorganized
aggregations of human tissue that form after the injection of human
embryonic stem cells into immunocompromised mice.
The resultant differentiated progenitor cells or fully
differentiated cells of the present invention, preferably human
differentiated cells, have numerous therapeutic and diagnostic, and
basic research applications. Most specifically, such differentiated
cells may be used for cell transplantation therapies. Human
differentiated cells have application in the treatment of numerous
disease conditions.
The subject differentiated cells may be used to obtain any desired
differentiated cell type. Therapeutic usages of such differentiated
cells are unparalleled. For example, human hematopoietic stem cells
and hemangioblasts may be used to treat many diseases that
compromise the immune system, such as AIDS, cancer therapy, or
age-related immune dysfunction. Hematopoietic stem cells can be
obtained, e.g., by fusing adult somatic cells of a cancer or AIDS
patient, e.g., fibroblasts or blood cells with an enucleated
oocyte, obtaining inner cell mass cells, and culturing such cells
in ovo under conditions which favor differentiation until
hematopoietic stem cells or hemangioblasts are obtained. By way of
a non-limiting example, hES or hEDC cells or primitive mesodermal
cells derived from such cells can be injected in ovo using one of
the techniques described herein in conjunction with stromal
fibroblasts from the aorta-gonadal-mesonephros region of a
non-human mammalian embryo or fetus or avian species to induce the
differentiation of the cells into hemangioblasts and hematopoietic
stem cells. Such cells may then be used with or without genetic
modification for the treatment of diseases including AIDS, cancer,
and immune dysfunction. The cells can also be used in veterinary
practice to treat canine or feline disease using cell therapy.
Alternatively, adult somatic cells from a patient with a
neurological disorder may be fused with an enucleated oocyte, human
inner cell mass cells obtained therefrom, and such cells cultured
in ovo under differentiation conditions to produce neural cell
lines and neural progenitor cells lines. Specific diseases
treatable by transplantation of such human neural cells include, by
way of example, Parkinson's disease, Alzheimer's disease, ALS,
palsy, and spinal cord injury among others. In the specific case of
Parkinson's disease, it has been demonstrated that transplanted
fetal brain neural cells make the proper synapses with surrounding
cells and produce dopamine. This can result in long-term reversal
of Parkinson's disease symptoms and disease progression.
The great advantage of the subject invention is that it provides an
essentially limitless supply of isogenic or homozygous MHC cells
suitable for transplantation.
Therefore, it will obviate the significant problem associated with
current transplantation methods, i.e., rejection of the
transplanted tissue which may occur because of host-vs-graft or
graft-vs-host rejection. Conventionally, rejection is prevented or
reduced by the administration or anti-rejection drugs such as
cyclosponine. However, such drugs have significant adverse
side-effects, e.g., immunosuppression, carcinogenic properties, as
well as being costly. The present invention will eliminate, or in
the case of homozygous MHC cells, greatly reduce the need for
anti-rejection drugs.
In addition, the present invention provides a means of directly
differentiating cells in the context of a SPF culture system
capable of generating complex tissues. It also allows for the
introduction of inducer molecules and cells from similar or
identical SPF species to direct the differentiation of the cells
without the complication of pathogen transmission from murine or
other retroviruses or other unknown agents.
In addition, the present invention provides methods to culture
mammalian teratomas near the CAM of an embryonated telolecithal or
eutelolecithal egg such that the teratoma is provided vascular
support from the developing chick. Such a teratoma can be later
removed from the egg and cannulated to provide a growing and
vascularized three-dimensional tissue. Since many complex tissues
are limited by the rate of diffusion of gases such as oxygen and
carbon dioxide and the exchange of nutrients and waste products,
the ability to assemble three dimensional aggregates of cells
derived from such cells as hES and hEDC cells with vasculature is
an important and novel advance facilitating the production of such
tissues as renal tissue, heart tissue, liver tissue, pancreatic
tissue, lung, as well as many other tissue types with dimensions in
excess of 0.5 mm in diameter.
B. The Transfer and Development of Non-Human Mammalian Embryos In
Ova
In another embodiment of the invention, whole and intact non-human
mammalian embryos and fetuses are gestated in ova. This system
would have great utility in producing cloned offspring where the
relative inefficiencies and high cost of recipient animals leads to
a high end cost of product. Animals such as domestic pigs whose
extraembryonic membranes closely resemble that of the avian embryo
and whose placenta does not form a syncitia with the maternal
uterus are especially suited for development in ovo. In addition to
providing a means of gestating domestic animals, genetically
modified non-human animals developed in ovo provide a sterile and
SPF system for producing cells and tissues for xenotransplantation.
In addition, the non-human animal developing in ovo can be used as
an intact animal to induce the differentiation of mammalian
pluripotent stem cells including hES and hEDC cells. By way of
non-limiting example, hES or hEDC cells or primitive mesodermal
cells derived from such cells can be injected into the
aorta-gonadal-mesonephros region of a non-human mammalian embryo or
fetus to induce the differentiation of the cells into
hemangioblasts and hematopoietic stem cells.
C. The Transfer of Reprogrammed Bovine Chromatin into the
Blastodisc In Ovo
The high value placed on mature human oocytes will lead to improved
technologies to remodel the chromatin of human cells in oocyte
extracts, or eventually to reprogram human DNA using defined
molecular components. Such technology is currently known in the art
where the extract is obtained from metaphase II oocytes. The
reprogrammed chromatin resulting from such reprogramming can be
injected into the blastodisc of an unfertilized telolecithal or
eutelolecithal egg with resulting rounds of karyokinesis and
cytokinesis resulting in reconstituted and reprogrammed cells
within the vitelline membrane. Such cellular reconstitution,
especially where such cells can be subsequently grown and
differentiated in ovo as described in the present invention,
provides an efficient and cost-effective means of producing
differentiated cells of many kinds under SPF conditions and would
therefore have great utility and value in producing human and
non-human animal cells for basic research, drug discovery, and cell
therapy.
D. The Co-Culture of SPF Avian Cells and Human Pluripotent Stem
Cells In Vitro
The present difficulties of differentiating human pluripotent stem
cells, such as hES cells into desired differentiated cell types
such as definitive hemangioblasts, pancreatic islet cells, heart
muscle precursor cells, neural progenitor cells, renal cells, liver
cells, lung cells, cartilage cells, or dermal cells demonstrates
the need for new technologies to direct the differentiation of such
pluripotent cells and to grow the cells in a defined pathogen-free
culture system. In addition to providing new pathogen-free
differentiation conditions, the present invention provides a novel
mean of expanding hES cells in vitro with feeder cells that, unlike
murine embryo fibroblasts, are known to be pathogen free, thereby
allowing the hES cells to be cultured in conditions that assure
their being free of exogenous pathogens and therefore minimizing
the risk of transmitting pathogens to patients in need of such cell
therapy.
EXAMPLES
Example 1
Human Embryo-Derived Cells Differentiated in Juxtaposition to an
Embryonated Telolecithal Egg
Approximately 10.times.10.sup.6 human ES cells were trypsinized
from culture, the trypsin was neutralized with 10% FCS in DMEM and
the cells pelleted and resuspended in DMEM. Approximately
1.times.10.sup.6 human ES cells were injected within the vitelline
membrane of an embryonated SPF egg (Charles River) at two days of
incubation at 0.5 cm from the avian embryo. At day 15, the mass of
cells were identified beneath the yolk sac membrane, fixed, and
Hematoxylin-and-eosin stained. In this example the cells were fixed
with formaldehyde, however there are many fixative agents known to
those skilled in the art which could be used. As shown in FIG. 5,
dense sheets of cells ranging from vacuolated mesenchymal to round
cells were visible, consistent with a predisposition to teratoma
formation. Yolk sac associated epithelial cells were also
observed.
Example 2
Human Embryonic Stem Cell Lines Maintained in the Undifferentiated
State Using SPF-Chick Embryonic Feeder Cells
Preparation of CEF:
CEF were isolated from 7-8 day old chicken embryos with the heads
left on, using the previously described techniques for isolation of
mouse embryonic fibroblasts. Briefly, the embryos were eviscerated,
the heads left on, digested with trypsin and plated onto gelatin
coated plates in DMEM, supplemented with 10% FBS, glutamine and
penicillin-streptomycin. The cells were frozen at passage one and
used at passage 2 after mitotic inactivation with mitomycin C.
The hES cell lines, H9, H7 (both NIH-approved) and ACT-4 were
consecutively cultured on CEF for 3-6 passages without significant
changes in undifferentiated morphology or growth rate. Passages
used for the experiment: H-9 & H-7: H-9 started passage 38
through passage 40, H7 started 29 and through passage 35; and ACT 4
derived here from passage 9-11 and 15-19.
Expression of the markers of pluripotency (Oct-4, alkaline
phosphatase, SSEA3, SSEA-4, TRA-1-60, TRA-1-81) remained high in
hES cells (line H7) after culturing on CEF for 4 consecutive
passages. FIG. 6 shows the undifferentiated hES grown on the
CEF.
Example 3
Non-Human Embryonic Development within a SPF Avian Egg and the Use
of the Porcine Embryo to Direct the Differentiation of Human
Pluripotent Cells
A cloned or normal porcine blastocyst with or without a transgenic
suicide gene is held with an aspiration pipette under low
magnification and the trophectoderm is torn opposite the inner cell
mass to yield near-planar aggregation of cells. The torn blastocyst
is injected with a 200 micron pipette into an unfertilized but
fresh SPF windowed avian egg at or near the blastodisc. The
resulting reconstructed egg is then resealed with kitchen wrap as
is well known in the art and cultured at 37.degree. C. on a racking
platform. At the point when cell differentiation of a desired type
is occurring in the porcine embryo, hES or hED cells are injected
into the porcine embryo. In the case of hematopoietic
differentiation, the human pluripotent stem cells are injected into
the aortic-gonadal-mesonephros region of the porcine embryo to
induce differentiation into hematopoietic differentiation such as
hemangioblasts.
Example 4
The Use of SPF Avian Mesodermal Cells of the
Aorta-Gonadal-Mesonephros Region to Direct the Differentiation of
Human Pluripotent Cells into Hemangioblasts
hES or hED cells are co-cultured with mesenchymal cells dissected
from the aortic-gonadal-mesonephros region of SPF avian embryos to
induce differentiation into hematopoietic differentiation such as
hemangioblasts. The co-culture is incubated in pathogen-free tissue
culture until primitive hemangioblasts are produced which are
subsequently purified by the use of antigens such as CD4, AC133,
c-kit, or other antigens well known in the art.
OTHER EMBODIMENTS
From the foregoing description, it will be apparent that variations
and modifications may be made to the invention described herein to
adopt it to various usages and conditions. Such embodiments are
also within the scope of the following claims.
The above specification, examples, and data provide a complete
description of the manufacture and use of the invention.
* * * * *
References